DNA microarray technology has been applied to many areas such as gene expression and discovery, mutation detection, allelic and evolutionary sequence comparison, genome mapping and more. Unfortunately, most applications fail to tap into the full capacity of microarray technology as many hybridization assays involve far less probes than are available using the full capability of the number of features possible in a high density microarray.
The advent of DNA microarray technology makes it possible to build an array of hundreds of thousands of DNA sequences in a very small area, such as the size of a microscopic slide. See, e.g., U.S. Pat. No. 6,375,903 and U.S. Pat. No. 5,143,854, each of which is hereby incorporated by reference in its entirety. The disclosure of U.S. Pat. No. 6,375,903 enables the construction of so-called maskless array synthesizer (MAS) instruments in which light is used to direct synthesis of the DNA sequences, the light direction being performed using a digital micromirror device (DMD). Using an MAS instrument, the selection of DNA sequences to be constructed in the microarray is under software control so that individually customized arrays can be built to order. In general, MAS-based DNA microarray synthesis technology allows for the parallel synthesis of over 800,000 unique oligonucleotide features in a very small area on a standard microscope slide.
Many applications require looking at markers across the entire human genome. The genome is typically too complex to be studied as a whole, and thus techniques must be used to reduce the complexity of the genome. A common technique that is often used is to amplify regions of the genome by Polymerase Chain Reaction (PCR).
This, however, requires that each section of the genome be amplified in either individual or multiple PCR reactions also known as multiplex PCR in a reaction tube. Conducting multiplex PCR in a tube, however, is limited to at most a few hundred reactions. Each primer pair for each reaction must be synthesized serially, quality controlled, and cataloged. Each unique primer must then be added to each reaction in the appropriate combination. This adds significant expense and time for each multiplex PCR reaction. Thus, it would be advantageous to be able to synthesize a plurality (i.e., thousands to tens of thousands) of DNA probes on an array to amplify a plurality of DNA target sequences present in a sample.
The DNA microarrays are generally synthesized by using light to direct which oligonucleotides are synthesized at specific locations or features on an array. Typically, only one nucleotide sequence is synthesized at each feature of the array, even though there are multiple DNA probes in each feature. This is because all the DNA probes in each feature have the same nucleotide sequence. However, in order to perform PCR on the array, two different oligonucleotide sequences must be synthesized in the same array feature. Accordingly, methods for amplifying DNA on a microarray are disclosed.